1. Field of the Invention
The present invention relates to E. coli microorganisms carrying recombinant plasmids constructed in vitro and their use for producing L-tryptophan by fermentation.
2. Brief Description of the Prior Art
The production of L-tryptophan from carbohydrates in wild type microorganism strains, has been obtained in the prior art through artificial mutants therefrom. Among the known examples of such artificial mutants are those of the genera Brevibacterium resistant to 5-methyl-tryptophan (U.S. Pat. No. 3,700,539), Bacillus resistant to 5-fluorotryptophan (Japanese Published Unexamined Patent Application Number 20391/1974), and Enterobacter resistant to 5-methyl-tryptophan (Japanese Published Unexamined Patent Application Number 57888/1976).
The most efficient known microorganism to produce tryptophan is Corynebacterium glutamicum ATCC 21851, which requires phenyl-alanine and tyrosine, and is resistant to 4-methyl-tryptophan, 6-fluoro-tryptophan, 4-amino-phenylalanine, 4-fluoro-phenylalanine, tyrosine-hydroxamate, and phenylalanine-hydroxamate. This strain produced 16.8 mg/ml tryptophan from 15 g/dl of sugar derived from cane blackstrap molasses. However, the yield of tryptophan in this best known method, is still insufficient to fulfill commercial requirements.
The possibility of utilizing recently developed genetic recombination techniques, to engineer a microorganism capable of producing high levels of tryptophan is appealing. The general techniques for the introduction of genes, and the amplification in bacteria capable of expressing them have recently been described by Gilbert and Villa-Komaroff in Scientific American, 242: 74-94 (1980). Briefly, one or more genes from a donor organism, such as a prokaryotic or eukaryotic cell are introduced into a vector or plasmid (extrachromosomal circular DNA) in vitro, by means of a splicing/ligation sequence using endonuclease and ligase enzymes respectively. The hybrid plasmid containing the gene or genes is then mixed with cells of a host organism, usually a prokaryotic bacterial microorganism. A dilute solution of calcium chloride renders the bacteria permeable and the cells will take up plasmids from solution. Reproduction of the plasmid-carrying host microorganisms then produces millions of identical copies of the recombinant DNA. If the appropriate genetic control sequences are present, the amplified gene or genes will produce corresponding enzymes using the available protein-synthesizing apparatus of the host.
Hershfield et al, for example (Proceedings of the National Academy of Sciences, USA, 71: 3455-3459 (1974)), have reported the insertion of a DNA fragment of E. coli possessing genetic information related to tryptophan (trp) production (trpA-E gene), into the Col El (colicinogenic factor El) plasmid. When a tryptophan auxotroph of E. coli was transformed with the resulting hybrid plasmid (Col El trp), the tryptophan auxotroph became a tryptophan prototroph. Elevated levels of tryptophan biosynthetic enzymes were reported. These authors however, did not further attempt to maximize tryptophan production, as their aim was solely to demonstrate the utility of the Col El plasmid as a molecular vehicle for cloning and amplification of DNA.
The biosynthetic pathways for the synthesis of aromatic amino acids (tryptophan, tyrosine, phenylalanine) in bacteria, are shown in Chart I: ##STR1## Tyrosine, phenylalanine and tryptophan are produced from a common biosynthetic intermediate, chorismic acid. Tyrosine and phenylalanine are furthermore derived from another common intermediate, prephenic acid. The first major enzyme of the tryptophan pathway is anthranilate synthetase which, in E. coli is subject to feedback inhibition by L-tryptophan. The degradation of tryptophan into indole by the enzyme tryptophanase is also shown in Chart I.
A need continues to exist for microorganisms capable of producing high levels of tryptophan. A need also continues to exist for a method for the production of tryptophan in high yields, using a microorganism distinct from those obtained by the mutation techniques of the prior art.